Information concerning the ratio(s) of concentration or amount of chemical compounds, including biological molecules such as proteins, glycoproteins, nucleotides, and the like, in a multi-component mixture, extract, system, etc., or the ratio of the amount of sub-populations of such biological molecules, is important in order to identify and characterize the mixture, extract, and/or system. This information is useful for the characterization of raw materials and/or end products in manufacturing of biologicals and biopharmaceutical agents; in-process monitoring of biotechnology products; quality control of multi-component biopharmaceuticals, such as vaccines, for diagnosis, prognosis, and monitoring of diseases and response to treatment, for non-morphological evaluation of tissues and body fluids in pathological analysis, etc. This information is also important in toxicology, forensic medicine, drug safety studies, and other fields of biomedicine.
Hereafter, the word biopolymer should be taken as a descriptive word for compounds of biological origin, such as proteins, enzymes, nucleic acids, polynucleotides, peptides and the like, and derivatives of such compounds. Analysis of a mixture of chemical compounds and/or biopolymers is generally based on separation of the mixture into its individual components. The separation process immediately precedes or is concurrent with the measurement of the level of the individual components. Separation procedures currently used in analytical determinations include liquid chromatography, electrophoresis, electrofocusing, and the like. These procedures are generally time- and labor-consuming, expensive, and are customarily performed by highly-skilled personnel.
Sometimes only the ratio of the amounts of two biopolymers or sub-populations of different molecular forms of the same biopolymer in a multi-component system is of interest. Several distinct forms of a protein are often present in a biological fluid such as blood, urine, etc. These forms may differ by a number of certain residues, such as sialic acids or glucose residues, by glycosylation pattern, by occurrence as free individual molecules vs. complexes with certain ligands. A biopolymer simultaneously existing in different molecular forms may be viewed, for the purpose of this invention, as a population consisting of two or more sets of sub-populations. The ratio of the amounts of these sub-populations is often important for medical diagnostics, quality control, and other applications.
In certain applications, including medical diagnostics, it is often useful to compare a population of biomolecules to a reference population, or to compare a sub-population to a reference sub-population. Hereinafter both cases are referred to as sub-population. In this context, the ratio of the amounts of sub-populations refers to a quantitative measure of the difference between a sample comprising of a sub-population(s) and a reference sample. For these applications it is also possible to use a particular numerical value of the ratio as defined above, as a numerical cut-off value to delineate when a sample comprising of a sub-population(s) of biomolecules could be considered clinically different from a reference sample. Monitoring a change in the ratios of components and/or sub-populations may be of diagnostic value even when information regarding the exact nature of changes in the components and/or subpopulation is not available.
An illustrative example of how the determination of the ratio of the amounts of sub-populations is useful is offered by carbohydrate-deficient transferrin (CDT) detection.
Found in blood serum, modified forms (isoforms) of transferrin (Tf) are proposed as a marker of long-term alcohol abuse. Changes in the structure of the carbohydrate part of the protein Tf, such that the sialic acid content is decreased, are presumed to be indicative of long-term alcohol abuse. Tf is found in the blood as a population comprised of several forms which are different in regard to their sialic acid content and pI value. CDT is also found in the blood to exist as a sub-population of several modified isoforms of Tf, including isoforms with low sialic acid content. The relative amount of these isoforms is increased under alcohol abuse. The ratio of CDT to total Tf that may be indicative of alcohol abuse refers to the relative amount of the sub-population of modified Tf with low sialic acid content to the total amount of all isoforms of Tf.
A second example of how the determination of the ratio of the amounts of sub-populations is useful is by detection and determination of prostate-specific antigen (PSA). Recently, the diagnostic power of the prostate-specific antigen (PSA) analysis has been found to significantly increase when one examines the ratio of the free PSA content in serum to that of the PSA complex with anti-.alpha..sub.1 -chymotrypsin.
A third example is provided by analysis of the ratio of glycated hemoglobin to total hemoglobin content as an important indicator of the long-term status of diabetes. Since glycated hemoglobin may refer to a sub-population comprising different molecular forms of hemoglobin, it is the ratio of the amount of that sub-population to the total amount of hemoglobin (total population of all isoforms) in the mixture that is of clinical importance.
In the three examples discussed above it is possible to use the ratio obtained in the present invention to detect a departure of the sub-population from a reference value that is of specific clinical diagnostic value. The utility of the ratio obtained using the present invention would then be to determine, typically using a cut-off value, whether a sample is clinically different from the reference. The level of difference could be delineated from the quantitative value of the ratio between the two samples.
Measurements of all these ratios are generally based on separation of the diagnostically-relevant protein isoforms. An analytical method capable of detecting the ratios in question without first separating the isoforms should be more efficient than known separation-based procedures.